Genetic disorders are pathologies caused by macroscopic alteration in chromosomes or microscopic lesions (point mutations, deletions or insertion) in genes. A genetic disorder is heritable when the genomic alteration is present in progenitors and can be transferred to the progeny. A genetic disorder is autosomal dominant when only one mutated copy of the gene (inherited by one progenitor) is necessary and sufficient for an organism to be affected, A genetic disorder is autosomal recessive when two copies of the gene (inherited by both progenitors) must be mutated for an organism to be affected. Over 4000 human diseases are due to single gene mutations, many of them affecting striated muscle tissues. Among these, sarcoglycanopathies are severe muscular dystrophies caused mainly by missense mutations in either α-, β-, γ- or δ sarcoglycan coding genes and are identified as Limb Girdle Muscular Dystrophy type 2D, 2E, 2C and 2F, respectively (Laval and Bushby 2004). Sarcoglycans (SGs) form a tetrameric complex linked to the dystrophin-associated protein complex and, in addition to the main structural role, they are involved in signaling [Barton 2006, Yoshida et al 1998]. In particular, α-SG is an ecto-ATPase enzyme [Sandonà et al 2004] possibly implicated in the extracellular ATP-dependent modulation of skeletal muscle contractility [Sandonà et al 2005]. Gene defects in a single sarcoglycan result in the absence or reduced expression of all SG subunits, with impaired tetramer formation and plasma membrane localization [Sandonà and Betto 2009]. About 75% of α-SG, 59% of β-SG, 40% of γ-SG and 57% of δ-SG genetic defects are missense mutations, known to generate full length proteins with single aminoacid substitution [Leiden Open Variation database]. Recently, it has been demonstrated that α-SG missense mutants are substrates of the ER quality control and are prematurely disposed of by the ubiquitin-proteasome system [Gastaldello et al 2008, Bartoli et al 2008].
In 1969, Brody first described in a human patient a muscular disorder characterized by an “exercise-induced impairment of muscle relaxation”: muscle contraction was normal but relaxation appeared delayed after repetitive contractions [Brody 1969]. So far, Brody's disease (BD) is known as a rare inherited disorder of skeletal muscle due to a sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) deficiency, resulting from missense, non sense mutations and in frame deletions of ATP2A1 gene, coding for SERCA1 isoform [Bertchtold et al 2000].
Three isoforms of SERCA proteins are differentially expressed by three genes. The SERCA1 isoform is expressed in fast-twitch (type 2) skeletal muscle. SERCA1 deficiency results in delayed muscle relaxation due to prolonged increase of calcium concentration in skeletal muscle fibres cytoplasm.
A muscular disorder defined as “congenital pseudomyotonia” (PMT) [Testoni et al 2008] has been described in bovine species. Clinical symptoms are exercise-induced muscle stiffness. DNA sequencing provided evidence of a missense mutations (R164H) in bovine ATP2A1 gene [Drögemüller et al 2008]. Moreover, biochemical results clearly demonstrated that cattle pathological muscles are characterized by a selective reduction in the expression level of SERCA1 protein, which accounts for the reduced Ca2+-ATPase activity. By contrast, SERCA1 mRNA levels found in all affected animals were comparable with mRNA expression in normal samples [Sacchetto et al 2009].
For both Brody disease and cattle PMT a defect of ATP2A1 gene, resulting in selective reduction in SERCA1 expression level, has been indicated as causative of the disease and cattle PMT has been defined as the true counterpart of human Brody disease.
Since the mutations of ATP2A1 gene do not affect the transcription [Sacchetto et al 2009], it has been hypothesized that the resulting protein could be corrupted and could have an enhanced susceptibility to the protein degradation via ubiquitin-proteasomal pathway before being embedded into Sarcoplasmic Reticulum (SR) bilayer. This hypothesis turned out to be correct: results have demonstrated that SERCA1 R164H mutant is substrate of the quality control system and prematurely disposed of by the ubiquitin-proteasome pathway (Bianchini et al submitted for publication).
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited, potentially fatal, arrhythmogenic disease characterized by stress- and/or emotion-induced life-threatening cardiac arrhythmias [Liu et al 2008]. Mutations in the cardiac ryanodine receptor (RyR2) gene have been associated with the autosomal dominant form of CPVT.1 whereas the autosomal recessive form of CPVT has been linked to mutations in CASQ2 and, recently, TRDN genes, encoding calsequestrin2 and triadin, respectively [Beard et al 2004; Roux-Buisson et al. 2012]. These proteins, together with RyR2 and junctin, form a quaternary macromolecular complex at the junctional SR of cardiomyocytes responsible for SR Ca2+ release during cardiac muscle contraction. Investigations performed in knock-in models of dominant CPVT and recessive CPVT have demonstrated that abnormal Ca2+ release induces cell-wide Ca2+ waves, delayed after depolarizations and triggered activity, all of which lead to arrhythmogenesis [Liu et al 2009]. In the knock-in mouse model (CASQ2R33Q/R33Q), drastic reduction of CASQ2 is accompanied by decrease of triadin (25%) and junctin (70%) without any change of the relative transcripts [Rizzi et al 2008]. It has been demonstrated that the strong reduction of the D307H CASQ2 mutant, also linked to the recessive form of human CPVT, is due to the activity of the ubiquitin-proteasome system. In the knock in mouse model CASQ2D307H/D307H, the cardiac arrhythmia developed as consequence of CASQ2 mutant degradation, can be counteracted by systemic administration of the proteasome inhibitor Velcade that allows partial rescue of the mutant CSAQ2, i.e., 20% increase over not treated mice [Katz G et al 2013]. Similar results have been obtained by our group in the CASQ2R33Q/R33Q knock-in mouse by systemic delivery of Velcade (unpublished data). The T59R triadin mutant, recently identified in a CPVT patient, has been studied in both cellular and animal models suggesting it is prematurely disposed of by the ubiquitin-proteasome system of the cell [Roux-Buisson N et al. 2012].
Sarcoglycanopathies, BD and CPVT, even though affecting striated muscle, are very different genetic disorders both for symptoms and etiology. However, it is possible to recognize as common trait of these disorders the posttranscriptional removal of the mutated gene product because of folding problems, that leads to a de facto loss of function.
At present, there are no effective treatments for sarcoglycanopathies, Brody's disease (BD) or the recessive forms of Cathecolaminergic Polymorphic Ventricular Tachycardia (CPVT).
Recessive CPVT, for example, shows an incomplete response to β-blockers, that results in the recurrence of ventricular arrhythmias and cardiac arrest [Hayashi et al 2009]. BD patients are usually treated with dantrolene [Vattemi et al. 2010], a muscle relaxant (blocker of the dihydro-pyridine receptor-RyR complex), but due to liver toxicity, dantrolene is unsuitable for long-term treatment. Both gene and cell therapy strategies are under evaluation for the cure of sarcoglycanopathies and CPVT, but are far from being amenable of clinical trial [Daniel et al 2007; Denegri et al 2012]. Exon skipping strategy, very promising in Duchenne Muscular Dystrophy [Hoffman et al 2011] is not appropriate for sarcoglycanopathies, CPVT and BD since mutant proteins don't have dispensable sequence that could be skipped away. The use of molecules able to promote stop-codon-read-through is potentially applicable in these disorders when a nonsense mutation is present. However, in sarcoglycanopathies, for example, the percentage of missense mutations is considerably higher than that of other gene defects.
There are no drugs currently approved to treat sarcoglycanopathies, Brody's disease (BD) and the recessive forms of Cathecolaminergic Polymorphic Ventricular Tachycardia (CPVT) and therefore there is a great unmet need for the treatment of such diseases.
We have now found that small molecules known as “CFTR correctors” are able to reverse the pathological phenotype of sarcoglycanopathies BD and CPVT by promoting folding and proper targeting of the mutated misfolded proteins and thus can be used in the treatment of genetic disorders affecting striated muscle selected from sarcoglycanopathies, Brody's disease (BD) and the recessive forms of Cathecolaminergic Polymorphic Ventricular Tachycardia (CPVT).